1. Volume 26, Issue 5, Pages 496-510 (September 2013)
A Conserved RhoGAP Limits M Phase Contractility and Coordinates with Microtubule Asters to Confine RhoA during Cytokinesis 
Esther Zanin, Arshad Desai, Ina Poser, Yusuke Toyoda, Cordula Andree, Claudia Moebius, Marc Bickle, Barbara Conradt, Alisa Piekny, Karen Oegema 
Developmental Cell 
Volume 26, Issue 5, Pages (September 2013)
DOI: /j.devcel
Copyright © 2013 Elsevier Inc. Terms and Conditions

2. Developmental Cell 2013 26, 496-510DOI: (10.1016/j.devcel.2013.08.005)
Copyright © 2013 Elsevier Inc. Terms and Conditions

3. Figure 1
The GAP Activity of RGA-3/4 Restrains RhoA Activity during Mitosis and Cytokinesis
(A) Schematics of a wild-type embryo (left), an ECT-2-depleted embryo (middle), and of the predicted effect of inhibiting the RhoA GAP (right).
(B) Of the 23 RhoGAPs in C. elegans, only three (CYK-4, RGA-3, and RGA-4) are embryonic lethal (Table S1).
(C) Left: schematics of CYK-4 and the cyk-4::gfp transgene. Right: immunoblot of worm extracts from the indicated transgenic strains probed for GFP and α-tubulin as a loading control.
(D) Left: schematics of RGA-3 and RGA-4 and of the gfp::rga-3 transgene. Right: immunoblot of worm extracts from N2 control and indicated transgenic strains; the strain expressing GFP::RGA-3 was homozygous for rga-3 and rga-4 deletions (rga-3/4Δ), whereas the strain expressing GFP::RGA-3GD was not.
(E) Differential interference contrast images during early and late cytokinesis for the indicated conditions.
(F) Graph plotting embryonic lethality, first division cytokinesis failure, and hypercontractility (>2 ingressions during mitosis). n = number of embryos.
(G) Immunofluorescence of one-cell stage wild-type (left) and rho-1(RNAi) (right) embryos stained with antibodies to RGA-3/4 (n > 12 for each condition).
(H) Central plane confocal images of one-cell stage metaphase rga-3/4Δ embryos expressing GFP-RGA-3GD after RNAi of the indicated genes (n > 7 embryos for each condition). Scale bars represent 10 μm.
See also Figure S1, Table S1, and Movies S1, S2, and S3.
Developmental Cell  , DOI: ( /j.devcel )
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4. Figure 2
Identification of a Human GAP that Opposes RhoA Activation during Mitosis
(A–C) HeLa cells were induced to express wild-type or constitutively active (Q63L) GFP::RhoA for 16 hr prior to imaging. (A) Bright field images. Scale bar represents 10 μm. (B and C) Graphs plotting the percentage of mitotic cells expressing each RhoA variant that had large protrusions (B) or exhibited cytokinesis failure (C). Error bars are SD from two or three independent experiments. n = total number of cells.
(D) Graph plotting the mean percentage of mitotic cells with large protrusions across two (38 genes) or three (33 genes) independent experiments following knockdown of each of the predicted human RhoGAPs. Mitotic cells from all four siRNAs targeting each gene were pooled. Error bars represent SD.
(E) Schematics of three human RhoGAPs with a domain architecture similar to C. elegans RGA-3.
(F) Images of unsynchronized HeLa cells stained for DNA and RhoA after treatment with a control siRNA or pooled siRNAs targeting the indicated GAPs. Arrowheads point to small blebs, which are present in controls; arrows point to large protrusions resulting from ARHGAP11A knockdown. Scale bar represents 10 μm.
(G) Phylogenetic tree of a GAP domain alignment highlighting conservation of the MP-GAP family in invertebrates and primitive metazoans (for accession numbers see Figure S2D).
See also Figure S2.
Developmental Cell  , DOI: ( /j.devcel )
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5. Figure 3
MP-GAP Controls Cortical Dynamics during Mitosis and Cytokinesis
(A) HeLa cells expressing BAC-integrated MP-GAP::GFP fixed at different cell cycle stages and stained for DNA, GFP, and microtubules.
(B) Immunoblot of HeLa cells expressing BAC-integrated MP-GAP::GFP treated with MP-GAP siRNA#1 or a control siRNA for 48 hr. Serial dilutions of control cells were loaded to quantify depletion level. Alpha-tubulin served as a loading control.
(C) HeLa cells expressing BAC-integrated Anillin::GFP filmed starting 48 hr after transfection with control or MP-GAP siRNA. Representative bright field (top) and confocal fluorescence (bottom) images are shown.
(D) Graph of the percentage of cells with large protrusions in interphase (n = 2 experiments), prometaphase/metaphase (n = 7 experiments), and anaphase (n = 7 experiments). Error bars represent SD. Total number of cells analyzed: control siRNA (55 interphase, 245 prometaphase/metaphase, 263 anaphase), MP-GAP siRNA (55 interphase, 140 prometaphase/metaphase, 140 anaphase).
(E) Graph showing the spatial distribution of protrusions in early anaphase fixed cells stained for RhoA and DNA. (40 protrusions from two independent experiments).
(F) Fluorescence confocal images of HeLa cells expressing BAC-integrated Ect2::GFP 48 hr after transfection with control or MP-GAP siRNA. Times are minutes after anaphase onset.
(G) Representative brightfield and confocal fluorescence images from time-lapse sequences of HeLa cells expressing Membrane::GFP (MyrPalm:mEGFP) or Ect2::GFP and treated with control or MP-GAP siRNA as indicated. Imaging was initiated ∼8 hr after release from a thymidine block. Times are minutes after anaphase onset. Graph shows percentage of early and late cytokinesis failure for each condition. Error bars represent SD of two (Membrane::GFP) or three (Ect2::GFP) independent experiments. The total number of cells analyzed was: Membrane::GFP (95 control siRNA, 90 MP-GAP siRNA); Ect2::GFP (179 control siRNA, 159 MP-GAP siRNA). Scale bars represent 10 μm.
See also Figure S3 and Supplemental Experimental Procedures.
Developmental Cell  , DOI: ( /j.devcel )
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6. Figure 4
MP-GAP Suppresses Cortical Hypercontractility by Targeting RhoA
(A) Immunoblot of stable isogenic HeLa cell lines expressing GFP::MP-GAP or GAP-deficient GFP::MP-GAPGD probed with antibodies to GFP and α-tubulin.
(B) Left: immunofluorescence images of a control cell line or cell lines expressing GFP::MP-GAP or GFP::MP-GAPGD after transfection with MP-GAP siRNA. Cells were stained for DNA, RhoA, and GFP. Right: graph showing the mean percentage of mitotic cells with protrusions. Error bars represent SD of three independent experiments. Total number of cells analyzed: control siRNA (170 No transgene), MP-GAP siRNA (158 No transgene; 170 GFP::MP-GAP; 158 GFP::MP-GAPGD).
(C) Left: Coomassie-stained gel of purified GAP domain used in the in vitro GAP assay. Right: graph showing production of free phosphate, monitored by absorbance at 650 nm, after incubation of purified MP-GAP domain or control buffer with RhoA, Rac1, or Cdc42. Numbers are the mean from three independent experiments; error bars represent SD.
(D) Right: immunofluorescence images of synchronized HeLa cells treated with control or MP-GAP siRNA, arrested in metaphase with MG132, and fixed and stained for DNA and RhoA. Left: graph of the maximum RhoA fluorescence intensity on the cell cortex (control and MP-GAP siRNA) or in the protrusions (MP-GAP siRNA) measured from 20 pixel wide linescans. The total number of linescans was: control siRNA (cortex, n = 48), MP-GAP siRNA (cortex, n = 64; protrusions, n = 50). P values are from Student’s t tests. Error bars represent SEM.
(E–H) Representative images of HeLa cells fixed and stained for DNA and RhoA. Graphs display the mean percentage of anaphase cells with protrusions. Error bars represent SD. Treatment protocols for each inhibitor/siRNA are outlined in Figures S4A–S4E. (E) HeLa cells treated for 6 hr with the RhoA inhibitor C3; the total number of analyzed cells in two independent experiments was control siRNA (30 DMEM; 37 C3), MP-GAP siRNA (49 DMEM; 46 C3). (F) HeLa cells transfected with control or MP-GAP siRNA for 24 hr followed by a second siRNA transfection (control or Ect2 siRNA) for 24 hr. The total number of analyzed cells in three independent experiments was: first siRNA control (second siRNA: 64 control; 67 Ect2); first siRNA MP-GAP (second siRNA: 69 control; 69 Ect2). (G) HeLa cells synchronized and treated with the PLK1 inhibitor BI2536 during anaphase. The total number of cells analyzed in two independent experiments was: control siRNA (114 DMSO; 84 BI2536), MP-GAP siRNA (130 DMSO; 151 BI2536). (H) HeLa cells treated for 1 hr with the Rho kinase inhibitor Y The total number of analyzed cells in two independent experiments was control siRNA (34 DMSO; 37 Y27623), MP-GAP siRNA (61 DMSO; 33 Y27623). Scale bars represent 10 μm.
See also Figure S4.
Developmental Cell  , DOI: ( /j.devcel )
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7. Figure 5
MP-GAP Inhibition Accelerates RhoA Accumulation but Does Not Alter RhoA Zone Dimensions along the Anaphase Spindle
(A) Schematic of the RhoA flux model.
(B) Time-lapse confocal fluorescence images of Anillin::GFP HeLa cells transfected with control or MP-GAP siRNA.
(C) Left: central plane confocal image and schematic illustrating the method used to measure the width of the Anillin::GFP zone. Right: graph plotting the width of the Anillin::GFP zone in cells transfected with control or MP-GAP siRNA. Data from two independent experiments were pooled. n = number of linescans analyzed. Error bars represent SEM.
(D) Mean Anillin::GFP fluorescence intensity in an 8 μm wide equatorial region 3, 6, and 9 min after the last metaphase frame. Mean fluorescence intensities were measured from cortical linescans as described in (C) and were normalized to the mean intensity in controls at 9 min (set to 100%). Data from two independent experiments were pooled. The total number of cells analyzed was: control siRNA n = 21, MP-GAP siRNA n = 19. Error bars represent SEM p values are from Student’s t tests.
(E) Experimental scheme used to treat cells with blebbistatin at anaphase onset.
(F) Width of the RhoA zone in fixed early anaphase DMSO and blebbistatin-treated cells measured as described in (C). n = number of linescans analyzed. Error bars represent SEM. Scale bars represent 10 μm.
Developmental Cell  , DOI: ( /j.devcel )
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8. Figure 6
MP-GAP-Mediated RhoA Flux Specifies RhoA Zone Dimensions when the Centrosomal Microtubule Asters Are Absent
(A) Immunofluorescence images of control, MCAK siRNA, and low dose nocodazole-treated cells stained for DNA, PLK1, and α-tubulin.
(B) Measurement of midzone length from DNA fluorescence images of fixed early anaphase cells using a 50 pixel wide line scan and measuring the distance between the two peaks of DNA fluorescence intensity.
(C) Graph of midzone length for control, MCAK siRNA-treated, and low dose nocodazole-treated cells. n = number of cells scored. Error bars represent SEM.
(D) Anaphase immunofluorescence images of cells treated as indicated and stained for DNA and RhoA.
(E and F) Graphs plotting the width of the Anillin zone (E) and the RhoA zone (F) measured as described in Figure 5C. n = number of linescans analyzed. P values are the Student’s t test; error bars represent SEM.
(G and H) Images of HeLa cells expressing Anillin::GFP. Representative time-lapse sequences (G) or three images of cells ∼13.5 min after anaphase onset (H) are shown for each condition.
(I) Cortical Anillin::GFP fluorescence measured using a pole-to-pole linescan 13.5 min after anaphase onset. The average cortical fluorescence intensity at metaphase was subtracted from each value and the two linescans for each cell were averaged to generate each individual trace. Scale bars represent 10 μm.
See also Figure S5.
Developmental Cell  , DOI: ( /j.devcel )
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9. Figure 7
MP-GAP Controls RhoA Zone Formation in the Absence of the Centrosomal Asters
In control cells, RhoA zone dimensions are dominantly specified by the centrosomal asters. MP-GAP inhibition does not increase RhoA zone dimensions when the asters are present, but it does lead to the formation of large protrusions. In the absence of the centrosomal asters, MP-GAP inhibition broadens the equatorial RhoA zone indicating that RhoA zone dimensions are controlled by MP-GAP-mediated RhoA flux.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions